The invention pertains to the field of detection of the presence of a pathogenic organism in the environment and in tissues of susceptible individuals. In particular, the invention pertains to the detection of the presence of the causative organism of Bacterial Kidney Disease (BKD) of salmonids.
Bacterial Kidney Disease (BKD) is a disease of salmonids, such as salmon and trout species. BKD is a systemic illness that causes high mortality. The course of the disease is typically chronic, although acute outbreaks also occur. The disease was first reported in Scotland in 1931 and has since been reported in many countries around the world, including the United States, Canada, Chile, England, France, Germany, Iceland, Italy, Japan, Spain, Turkey, and in the Balkan peninsula.
BKD is a slowly progressing systemic illness in which the clinical signs typically are not evident until the disease is well established. External signs typically include exophthalmos and small closed blebs or open lesions. The unruptured blebs contain fluid composed of blood cells and necrotized tissue. In advanced cases, the lesions may coalesce and form large shallow ulcers. Internally, the kidneys are the organs most often affected. They become swollen and show discrete white areas that contain leucocytes, bacteria, and host cell debris. In advanced cases much kidney tissue is destroyed and hematopoietic and excretory functions are both affected. Hemorrhages occur in the body wall and testes, and peritoneal fluid is commonly present. The hind gut can be hemorrhagic and filled with white or yellow viscous fluid.
Death due to BKD most commonly is sporadic and occurs over a long period of time, although subacute outbreaks with 25 to 50% mortality occurring within a few weeks have been reported. Juvenile salmonids show the greatest susceptibility to infection with the level of susceptibility being highly variable between species. Inapparent carriers of the disease are common, as infected fish may carry the causative organism of the BKD for years without showing any clinical signs. The disease may only manifest itself clinically when such carrier fish are stressed.
The causative organism of BKD is a slow-growing gram positive bacterium, Renibacterium salmoninarum. This bacterium lives both extracellularly and intracellularly in the salmonid host and has been shown to survive and even to multiply within macrophages. This permits the bacterium to spread readily throughout the body. The intracellular location and the widespread distribution of R. salmoninarum within the body make the infection difficult to treat because antibiotics may not reach all of the locations where the organism resides. Antibiotics typically employed, such as erythromycin, are cleared rapidly by the fish and require administration at near toxic levels. Additionally, BKD disease is one of the few bacterial diseases that can be spread both horizontally and vertically. R. salmoninarum can be transmitted in the egg.
Because of the widespread systemic distribution and intracellular location of the BKD bacterium, the presence of latent carriers, the ability of the disease to be transmitted vertically, and the lack of an effective bacterin, BKD has proven to be very difficult to control. Therefore, because of the potential for BKD to devastate a population, control efforts are concentrated in preventing exposure of infected fish to the BKD organism. It is common practice to isolate salmon returning to hatcheries, segregate fertilized eggs until diagnostic test results are evaluated, test each female, cull eggs from females with test results that suggest the presence of the BKD organism, and segregate remaining progeny according to the test values of the females.
Presently available tests for the BKD organism present problems that have not as yet been overcome. The isolation and identification of R. salmoninarum in samples from clinically infected fish is relatively easy. However, the slow growth of the organism and the fact that R. salmoninarum is a fastidiously growing organism requiring prolonged incubation at 15xc2x0 C. to produce colonies renders such tests of little utility in practice. Thus, serological methods are more usually employed to confirm the presence of the bacterium.
Immunofluorescence antibody tests (FAT) have commonly been used for demonstrating the presence of R. salmoninarum in infected tissues. Screening of ovarian fluid from asymptomatic brook trout populations using FAT and ELISA found that twice as many positives were detected using a FAT method than by ELISA. However, FAT reproducibility is poor for very low levels of infection, resulting in some infections being missed.
ELISA is widely used for the detection of R. salmoninarum, and several commercial kits are available. The most common technique used is a double antibody sandwich ELISA method. Samples of tissue (fish kidneys) collected for examination must be kept cold or frozen after collections. Tissues from clinically and sub clinically infected fish have ELISA reactions that are clearly distinct from those of uninfected fish. Unfortunately, ELISA does not work well on ovarian or seminal fluid. Since the ELISA test is performed on kidney tissue, diagnosis is not possible until after stripping eggs from the females. This requires extra manpower and space in order to strip eggs from females that will test above the culling threshold and to keep eggs segregated by female until the ELISA results are evaluated. There is about a four (4) week delay from submitting samples to a lab and receiving results. While several ELISA methods are available, it is important to note that the various evaluations as to their sensitivity and specificity have not been uniformly carried out.
Polymerase Chain Reaction (PCR) has been used for the detection and identification of R. salmoninarum directly within ovarian fluid or individual eggs. Present techniques rely on detection of DNA segments of the gene coding for the antigenic p57 protein. The nucleotide sequence coding for this protein is available from GenBank at the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (NIH) and has been assigned Accession No. AF123888. These PCR methods are very sensitive and have a high degree of specificity. The PCR/ovarian fluid method does not detect significantly more positives than ELISA methods from the kidneys, but is more rapid as results are obtained within 1 to 2 days and, in contrast to ELISA which utilizes kidney tissues, is non-lethal as,ovarian test fluid may be obtained when stripping eggs. Other studies have found PCR to identify higher numbers of kidney tissue and ovarian fluid samples from commercially reared brood stock fish to be positive for R. salmoninarum than were identified by culture.
One potential problem with PCR diagnostic methods is that a PCR-positive sample may contain some proportion of dead R. salmoninarum with detectable levels of DNA, such as due to previous antibiotic therapy or fish that have conquered the disease. Other problems that occur when using standard PCR methods include incorrect interpretation of band presence or absence because of improper primer annealing or buffer concentration. Additionally, the number of cycles to run for correct band amplification can vary between the primers and the type of machine used. These problems are further exacerbated when attempts are made to quantify the level of bacteria present. For R. salmoninarum, this has proven even more difficult with the necessary use of nested PCR for detection of the pathogen, since the addition of extra primers and increased cycle numbers leads to an increase in the detection of false positive bands.
In recent years, PCR methods have been adapted to provide both for detection and for quantification of nucleic acid sequences in a sample. For example, see, Higuchi, U.S. Pat. No. 6,171,785, incorporated herein by reference. These methods employ forward and reverse primers as in standard PCR plus one or more additional nucleic acid sequences that hybridize to the nucleic acid that is to be amplified. This additional nucleic acid sequence, termed a xe2x80x9cprobexe2x80x9d, hybridizes to a portion of the nucleic acid to be amplified between the portions that hybridize to the two primers, and is labeled in such a way so that each successive PCR cycle causes a change in the probe or its label. This change in the probe or its label causes activation or accentuation of the label to a degree that is related to the number of additional copies of the amplified nucleic acid during each PCR cycle. By utilizing such methods, referred to as xe2x80x9creal-timexe2x80x9d PCR, cycle-by-cycle detection of increasing PCR product is achieved by combining thermal cycling with label detection.
Most commonly, the label for the probe is a fluorescent label which provides a fluorescent output signal. This may be achieved by providing a probe which is double-labeled with a flourescent reporter dye at one end, typically the 5xe2x80x2 end, and a quencher dye at the other, the 3xe2x80x2, end. When the probe is intact, the proximity of the quencher dye to the reporter dye suppresses the fluorescent output of the reporter dye. During each PCR cycle, the 5xe2x80x2 nuclease activity of a DNA polymerase cleaves the probe, which separates the reporter dye from the quencher dye. This separation results in increased fluorescent output of the reporter dye.
During PCR, if the target of interest is present in a sample, the probe will specifically anneal between the forward and reverse PCR primer sites. The nucleolytic activity of the DNA polymerase cleaves the probe between the reporter and the quencher dyes only if the probe hybridizes to the target molecule. The increase in fluorescence is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, non-specific amplification is not detected. Only amplified products that contain the sequence complementary to the probe are recognized by the presence of the fluorescent signal, thereby eliminating certain elements related to the analysis of false-positives. Additionally, one or more other enzymes may be utilized to help limit the amplification of carry over transcription products.
This type of quantitative PCR permits the normalization of pipetting errors and volume changes, which may be done by dividing the reporter fluorescence by a passive reference, contained within each reaction, to determine the normalized reporter signal for each individual reaction. Software may be used to analyze the cycle-to-cycle increase in fluorescence intensity and compare this data to standards in order to determine starting copy numbers for absolute quantification or to compare against other unknown samples for a comparison of relative quantity.
To date, no such real-time method of detection of R. salmoninarum infection has been reported. The existence of such a method would have a significant impact on the ability to control this disease.
In one embodiment, the invention is a method for detection of the causative organism of Bacterial Kidney Disease (BKD) in a sample using real-time polymerase chain reaction (PCR). According to this embodiment, a forward primer, a reverse primer, which primers anneal to a DNA sequence that is specific to the Renibacterium salmoninarum genome, a labeled probe, which probe anneals to the DNA at a site between the sites of annealing of the forward and reverse primers, a DNA polymerase, and the four deoxynucleotide bases A, T, C, and G are combined with a test sample to form a mixture. The mixture is taken through successive (PCR) cycles, wherein the probe contains a label that is activated or accentuated to a degree that is related to the number of additional copies of the amplified nucleic acid during each PCR cycle. The PCR cycles include the steps of adjustment to a temperature at which the DNA is separated into single strands, adjustment to a temperature at which the primers and the probe anneal to complementary sequences on the DNA, and adjustment to a temperature at which the polymerase binds and extends a complementary DNA strand from each primer.
In another embodiment, the invention is a method for detection of the causative organism of Bacterial Kidney Disease (BKD) in a sample using real-time polymerase chain reaction (PCR). According to this embodiment, RNA in the sample is isolated and reverse transcribed to produce a cDNA. The cDNA is combined with a forward primer, a reverse primer, which primers anneal to a DNA sequence that is specific to the Renibacterium salmoninarum genome, a labeled probe, which probe anneals to the DNA at a site between the sites of annealing of the forward and reverse primers, a DNA polymerase, and the four deoxynucleotide bases A, T, C, and G to form a mixture. The mixture is taken through successive (PCR) cycles, wherein the probe contains a label that is activated or accentuated to a degree that is related to the number of additional copies of the amplified nucleic acid during each PCR cycle. The PCR cycles include the steps of adjustment to a temperature at which the DNA is separated into single strands, adjustment to a temperature at which the primers and the probe anneal to complementary sequences on the DNA, and adjustment to a temperature at which the polymerase binds and extends a complementary DNA strand from each primer. This embodiment is especially preferred if is it desired to determine the viability of the organism in the sample.
In another embodiment, the invention is a primer that binds to a DNA sequence that is specific to the Renibacterium salmoninarum genome, which primer, when combined with the DNA, a second primer that binds to the DNA, a DNA polymerase, and the four deoxynucleotide bases A, T, C, and G, provides exponential expansion of copies of the DNA during successive rounds of PCR. Preferably, the DNA sequence is a portion of the genome of Renibacterium salmoninarum that codes for the p57 protein. In a preferred embodiment, the primer has the sequence 5xe2x80x2-CAACAGGGTGGTTATTCTGCTTTC-3xe2x80x2, designated as Seq. ID No. 1. In another preferred embodiment, the primer has the sequence 5xe2x80x2-CTATAAGAGCCACCAGCTGCAA-3xe2x80x2, designated as Seq. ID No. 2.
In another embodiment, the invention is a kit that is useful for the detection of Renibacterium salmoninarum, the causative organism of BKD, in a sample.